Veterinary drug albendazole inhibits root colonization and symbiotic function of the arbuscular mycorrhizal fungus Rhizophagus irregularis

Abstract Arbuscular mycorrhizal fungi (AMF) are plant symbionts that have a pivotal role in maintaining soil fertility and nutrient cycling. However, these microsymbionts may be exposed to organic pollutants like pesticides or veterinary drugs known to occur in agricultural soils. Anthelminthics are veterinary drugs that reach soils through the application of contaminated manures in agricultural settings. Their presence might threaten the function of AMF, considered as sensitive indicators of the toxicity of agrochemicals to the soil microbiota. We determined the impact of the anthelminthic compounds albendazole and ivermectin on the establishment and functionality of the symbiosis between the model-legume Lotus japonicus and the AMF Rhizophagus irregularis. Our analyses revealed negative effects of albendazole on the development and functionality of arbuscules, the symbiotic organelle of AMF, at a concentration of 0.75 μg g−1. The impairment of the symbiotic function was verified by the reduced expression of genes SbtM1, PT4 and AMT2;2 involved in arbuscules formation, P and N uptake, and the lower phosphorus shoot content detected in the albendazole-treated plants. Our results provide first evidence for the toxicity of albendazole on the colonization capacity and function of R. irregularis at concentrations that may occur in agricultural soils systematically amended with drug-containing manures.


Introduction
Soil micr oor ganisms ar e pi votal in ecosystem functioning; the y ar e involv ed in nutrient cycling (Fier er 2017 ), modulate soil structure (Rillig et al. 2015 ), degrade and detoxify pollutants (Fenner et al. 2013 ) and e v entuall y enga ge into symbiotic r elationships with plants enhancing plant growth and tolerance to biotic and abiotic str essors (Triv edi et al. 2020 ).Ho w e v er, their health and functioning could be compromised by chemicals, like pesticides and veterinary drugs that, intentionally or unintentionally respectively, r eac h soil.The potential toxicity of pesticides on soil micr oor ganisms has attracted much attention (Feld et al. 2015, Vasileiadis et al. 2018, Romdhane et al. 2019, Zhang et al. 2019 ) and certain tests, like the Organisation for Economic Co-operation and Development (OECD) N transformation tests, are used at risk assessment le v el (OECD 2000).Ho w e v er, se v er al publications hav e highlighted the need for advancements in the assessment of the toxicity of pesticides on soil micr oor ganisms, suggesting the introduction of novel and standardized molecular tools (Martin-Laurent et al. 2013, Karpouzas et al. 2022 ).In this frame, the European Food Safety Authority (EFSA) Panel on Plant Protection Products and their Residues (PPR), recommended adding tests with arbuscular mycorrhizal fungi (AMF) to the data r equir ements and risk assessment, besides retaining and advancing the N-transformation test (EFSA Panel on Plant Protection Products and their Residues 2017 ).
AMF ar e m utualistic symbionts that associate with most land plants and are considered a k e y group in soil systems .T hey pro-vide important ecosystem services by influencing the terrestrial C cycling (Parihar et al. 2020 ) and improving the soil fertility (Fall et al. 2022 ).The plant-AMF association is ancient and one of the best studied symbiotic relationships in nature.AM symbiosis provides the plant with mineral elements (mainly phosphorus and nitr ogen), impr ov es water absorption and enhances the plant tolerance to biotic and abiotic stresses (Smith andRead 2008 , Begum et al. 2019 ).The symbiotic organelle of the AM symbiosis, the arbuscule, is a highl y br anc hed tr ee-like structur e that forms within cells of the inner root cortex and facilitates the nutrient exchange between the plant cell and the fungus (P aszk owski 2006 ).Both phosphate and ammonium transporters have been identified to function in the arbuscule-containing cells and mediate the transfer of phosphorus and nitrogen from the fungus to the plant, like the phosphate transporter PT4 (Volpe et al. 2016 ) and the ammonium transporter AMT2;2 (Guether et al. 2009 ) in the model legume Lotus japonicus .
The toxicity of pesticides on AMF has been the subject of several in vitro (Buysens et al. 2015 ) and soil studies (Jin et al. 2013, Karpouzas et al. 2014 ), with effects ranging from negative to positiv e or neutr al (Ha ge-Ahmed et al. 2019 ).The potential inclusion of AMF into the list of EFSA for Ecological Risk Assessment biological indicators, arises the need for the optimization of the r ele v ant testing protocols (Sweeney et al. 2022 ).The current standardized method (r e vie wed and confirmed in 2020) uses an in vitro spore germination bioassay of the mycorrhizal fungus Glomus mosseae (ISO/TS 10832 : 2009).Ho w e v er, Mallmann et al. ( 2018 ) r ecommended a modification of the specific ISO test with the inclusion of other AMF species and test conditions r ele v ant to the climatic zone where the pesticide is going to be authorized.Although spore germination is an important trait of the pre-symbiotic phase, other parameters should also be considered since effects may occur at both asymbiotic and symbiotic stages .T he breadth of available methodologies to test pesticides against AMF, ranging from in vitro bioassays to field trials, is r e vie wed in Sweeney et al. ( 2022 ).
Beside pesticides, a number of ecotoxicological studies have explored the effects of pharmaceuticals, mostly of antibiotics, on soil micr oor ganisms (Ding and He 2010 ).For example, it was sho wn that tetrac ycline exhibited c hr onic inhibitory effects on the growth of nitrifying bacteria (Katipoglu-Yazan et al. 2015 ), while the regular application of tylosin, sulfamethazine and chlorotetracycline in soil induced changes in the diversity of nitrogen fixing bacteria in soybean r oots (Re v ellin et al. 2018 ).Such findings forced r esearc hers to call on an increased consideration of microbial endpoints in antibiotics environmental risk assessment (Brandt et al. 2015 ), which can be further extended to other pharmaceuticals r eac hing a gricultur al soils like veterinary drugs.Unlike veterinary antibiotics, the potential effects of anthelminthic veterinary drugs on soil micr oor ganisms, including k e ystone functional groups like AMF, remain unknown.It is now well-documented that anthelminthics, used to control infestations of pr oductiv e animals by gastr ointestinal nematodes (Kaplan 2020 ), are not metabolized in animal body and they are excreted in animal feces (Aksit et al. 2015 ).These are then used as manures in agricultural soils contributing to the dispersal of anthelmintic compounds in a gricultur al soils (Iglesias et al. 2018, Porto et al. 2021 ).Upon their transfer in agricultural soils, anthelminthics like benzimidazoles (albendazole) and macrocyclic lactones (ivermectin and eprinomectin) would interact with the soil biota and/or transfer to surface or groundwater systems (Sim et al. 2013 ) or taken up by plants (Mesa et al. 2020 ) imposing a threat for the environment and human health (Na vrátilo vá et al. 2021 ).Most of the studies to date have focused on the potential effects of anthelmintics on soil fauna (Verdú et al. 2018, Barrón-Bravo et al. 2020 ), while little, if anything, is known about their off-target effects on the soil microbiota.
In the current study, we tested the potential toxicity of two of the most widely used anthelmintic compounds globally, albendazole and ivermectin, on the establishment and functioning of the plant-AMF symbiosis.For our tests we used the model legume Lotus japonicus and the commercially available AM fungus Rhizophagus irregularis strain DAOM.Different concentrations of the tested compounds wer e a pplied to our system and the de v elopment and functionality of the symbiotic r elationship wer e monitor ed by microscopic, physiological and molecular analyses.Our results provide unpr ecedented e vidence for the toxic potential of the anthelmintic compound albendazole to the establishment and functioning of AMF-plant symbiosis.

Biological ma terial, inocula tion, and gro wth conditions
The plant used in our assays was the Lotus japonicus L. ecotype Gifu B-129 wild-type.It was inoculated with R. irregularis strain DAOM (Agronutrition).Lotus japonicus seeds were surface scarified, sterilized and k e pt in water for 12 h at 4 • C, then germinated for 10 days at 21 • C (16 h light, 8 h dark).Plant seedlings wer e tr ansferr ed to magenta boxes containing 360 g baked sand and treated with 60 ml Long-Ashton nutrient solution (SLA) (Hewitt 1952 ).Each magenta box contained three plants and 300 spores of the AMF inoculum (almost 100 spores per plant) inside single sandwiches composed of nitrocellulose discs (Giovannetti et al. 1993 ) (Fig. S1).
Plants wer e gr own at 24-25 • C (16 h light, 8 h dark) and harvested at four-or five-week post inoculation in experiment 2 and 1, respectiv el y.

Application of anthelminthics
Analytical standards of the anthelminthic compounds ivermectin (97% purity, Sigma-Aldrich, St Gallen Switzerland) and albendazole (98% purity, Tok y o Chemical Industry, Zwijndr ec ht, Belgium) were used in our study.The main physicochemical and environmental fate properties of the anthelminthic compounds tested ar e pr esented in Table S1.Dense solutions of both compounds in DMSO (10 mg ml −1 ) were initially prepared.These were further diluted in Long-Ashton nutrient solution (SLA) to pr epar e working solutions of each compound in the intended concentration range.The working solution was then applied as a single application at the start of the experiment to the baked sand in the magenta boxes.Contr ol tr eatments r eceiv ed SLA amended with DMSO but without any anthelminthic compounds.In all cases, the amount of DMSO in the working solutions was 0.1%.All experiments were performed in five biological replicates for each treatment.

Experiment 1-effects of veterinary drugs on AMF root colonization and P content of plants
We first tested the toxic effects of albendazole and ivermectin on the colonization of the plant root by AMF.L. japonicus plants were inoculated with the fungus R. irregularis and treated with different concentrations of either albendazole (0.5, 5, and 15 mg L −1 ) or ivermectin (0.5, 5, and 50 mg L −1 ), while a set of samples served as controls (see abo ve).T hese concentrations are within the levels reported in soils (Thiele-Bruhn 2003, Liebig et al. 2010 , Babi ć and Muta vdži ć Pa vlo vi ć 2013 , Bele w et al. 2021 ) and they corr espond to exposure scenarios expected to occur in a gricultur al soils amended r ar el y (0.5 mg L −1 corr esponding to nominal concentr ation in soil of 0.083 mg kg −1 ) or r egularl y (5 mg L −1 corresponding to nominal concentration in soil of 0.83 mg kg −1 ) with contaminated manure or used as dumping sites for contaminated manure (15 or 50 mg L −1 corresponding to nominal concentration in soil of 2.5 or 8.3 mg kg −1 , r espectiv el y).The percenta ge of mycorrhizal root colonization was determined five weeks later, as well as the phosphorus content of albendazole-treated plants, as described below.

Experiment 2-effects of veterinary drugs on the functioning of AMF
Based on the outcome of experiment 1, a second experiment was emplo y ed to verify via molecular means the inhibitory effect of albendazole on AMF functioning and shed light on the potential mechanisms of its toxicity on AMF.Similarly to the first experiment, L. japonicus plants were inoculated with the fungus R. irregularis and treated with different concentrations of albendazole (0.5 and 5 mg L −1 ).A set of samples were treated with SLA without any albendazole to serve as control.All plants were harvested at 4 weeks and the expression levels of three AM symbiosis marker genes was determined by RT-q-PCR.The transcript levels of SbtM1 (Takeda et al. 2009 ) are indicators of normal stage transition during arbuscule de v elopment, while the tr anscript le v els of PT4 (Volpe et al. 2016 ) and AMT2;2 (Guether et al. 2009 ) are used as indicators of the phosphate and ammonium import activity from the fungus to the plant, r espectiv el y.

Determination of the levels of albendazole in the sand
Albendazole was extracted from 5 g of magenta sand with 10 ml of acetonitrile through shaking for 1 h on a horizontal orbital shaker at 220 r/m.The samples were then centrifuged for 5 min at 7500 r/m, and the supernatant was collected.The sand was reextracted with a further 10 ml of acetonitrile .T he supernatant from the two extraction steps were combined and passed through 0.45-μm hydrophobic syringe filters (PTFE Syringe Filter) before HPLC analysis .T he concentration of albendazole in the sand was determined by HPLC analysis in samples taken at zero, two and four weeks after application.
HPLC analysis was performed in a Shimatzu HPLC-DAD system equipped with a Grace 297 Smart RP C18 (150 mm × 4.6 mm).
Albendazole was detected at 205 nm using a mobile phase of acetonitrile/ phosphoric acid solution (0.1%) at a 20/80 v/v ratio.The flow rate of the mobile phase was 1 ml min −1 .More details about HPLC conditions and the validation of the extraction method can be found in Lagos et al. ( 2022 ).

Quantification of mycorrhizal colonization
AMF-colonized r oots wer e stained with 5% ink in 5% acetic acid solution (Vierheilig et al. 1998 ) and the le v els of colonization were quantified by microscopic examination of slides.Each slide was pr epar ed fr om a sub-sample of the roots of the three plants grown per magenta box and contained almost 20 cm of root.The (%) percentage of arbuscule formation was estimated by the examination of at least 100 e ye pieces per slide.Fi v e slides wer e examined per tr eatment, corr esponding to fiv e biological r eplicates.

Phosphorus plant content measurement
Fifteen to twenty mg dried abov e-gr ound plant material from L. japonicus plants were heated to ash at 550 • C, and then the ash was solubilized with 1 ml of 67% HNO 3 .The shoot extract was diluted to six ml with distilled water.Total concentration of P was determined with a PG T60 UV/ VIS Spectrophotometer, at 880 nm wavelength, following the Murphy and Riley color reaction method.

RN A extr action and RT-q-PCR anal ysis of AM symbiosis marker genes
Total RN A w as extr acted fr om plant r oots by a modified Lithium Chloride-TRIzol LS (Thermo Fisher) protocol (Holt et al. 2015 ).RN A concentration w as determined using a microvolume spectr ophotometer (DNA/Pr otein Anal yse) (Quaw ell).RN A w as DN Ase treated using DNAse I (Thermo Scientific) according to manufacturer guidelines.cDN A w as pr epar ed using 300 ng of total RNA, an oligo dT primer and SuperScript II (Invitrogen) according to manufactur er guidelines.RT-q-PCRs r eactions wer e performed using KAPA SYBR ® FAST qPCR master mix (KAPABIOSYSTEMS) and 200 nM primer concentr ation.Le v els of tar get genes wer e normalized to le v els of two r efer ence genes: L. japonicus ATP SYNTHASE2 ( LjATP2 ) and L. japonicus PROTEIN PHOSPHATASE2a ( LjPP2a ).RT-q-PCR r eactions wer e executed in a BioRad CFX Connect lightcycler (BioRad).The LinRegPCR softw are w as used for the data analysis (Ruijter et al. 2009 ).All primers are listed in Table S2.

Sta tistical anal ysis
To identify the drug concentration that may affect the establishment and functionality of the symbiotic relationship, pairwise comparisons between drug-treated and drug-untreated (control) r oot samples wer e emplo y ed ( Figs 1 , 2 , and 3 ).Statistical analyses were performed by Student's t-tests .A significance le v el of 5% was applied.
To test if there is a dose effect of albendazole on AMF root colonization, the Pearson's correlation coefficient was measured (between albendazole concentration and % AMF root colonization).The correlation was found significant at the level of 0.05 and the value of the correlation coefficient r was −0.532 (negative correlation, P value of 0.019) (Fig. S2).
Considering the albendazole dissipation experiment (Fig. 4 ), in order to compare the albendazole concentration between differ ent tr eatments and time points, statistical analysis was performed by one-way ANOVA followed by a post-hoc Tuk e y's test.

Albendazole negati v ely affects AM root colonization and P uptake by plants
Application of albendazole solution with a concentration of 15 mg L −1 , corresponding to a measured concentration in sand of 2.49 μg g −1 , resulted in significantly reduced levels of mycorrhizal colonization compared to the control treatment (Fig. 1 A, Fig. S2).No significant differences were observed when solutions with lo w er albendazole concentr ations (0.5 and 5 mg L −1 corr esponding to measured concentrations of 0.055 and 0.765 μg g −1 in sand, r espectiv el y) wer e a pplied to our system (Fig. 1 A), howe v er, a negativ e dose effect of albendazole was detected on the AMF root colonization by measuring the Pearson's correlation coefficient (Fig. S2).Contr astingl y, a pplication of iv ermectin in the plant substrate did not have any effect on the mycorrhizal root colonization, e v en when a high concentration of 50 mg L −1 was tested (Fig. 1 B).The differential activity of albendazole and ivermectin a gainst AMF r oot colonization may be attributed to the different mode of action of the two compounds.Ivermectin may not impede AMF root colonization as it has been reported to act as an allosteric modulator of glutamate-gated chloride channels in nematodes and insects, and also, ion channels of the host central nervous system (Martin et al. 2021 ), a mode of action not relevant for AMF and/or their plant host.On the other hand, benzimidazoles , like albendazole , act by inhibiting the polymerization of β-tubulin during microtubule formation in mitosis (Ramírez et al. 2001 ), a fundamental biological mechanism in the microbial world and beyond.Although this is the first report regarding the toxicity of albendazole to off-target microorganisms like AMF, previous studies have highlighted the inhibitory effects of benzimidazoles like the fungicides benomyl and carbendazim on AMF.These compounds are strong inhibitors of AM fungal colonization (Sukarno et al. 1993, O'Connor et al. 2009 ) and P uptake (Larsen et al. 1996, Kling and Jakobsen 1997, Schweiger and Jakobsen 1998 ) with effects observed at agricultural relevant dose rates.
To find out whether albendazole has a direct effect on the plant-AMF symbiosis or the reduced mycorrhizal colonization phenotype results from a general effect of this compound to the plant de v elopment, we monitor ed the plant gr o wth b y measuring the shoot and root length.No differences were detected in either shoot or root length of the plants treated with increasing concentrations of albendazole (Table 1 ).This observation is in line with pr e vious findings, as albendazole did not affect the germination of mustard seeds (Prchal et al. 2016 ).
To further explore if the observed inhibitory effect of albendazole on AMF colonization translates into an impairment of phosphorus (P) uptake by plants, we monitored the P levels in the plant shoots.We noted that the plants treated with solutions of 5 or Table 1.Application of albendazole does not affect plant growth.

Shoot length (cm)
Root length (cm) Measur ements wer e performed fiv e weeks after the a pplication of differ ent concentrations of albendazole (or DMSO for the control treatment).
No significant difference was detected between treatments (n = 5).
15 mg L −1 albendazole (corresponding to 0.765 and 2.49 μg g −1 , r espectiv el y in sand) accumulate significantly lo w er levels of P in the shoot tissues, compared to control plants not receiving albendazole (Fig. 2 ).No significant changes were detected in the shoot P content when plant roots were treated with solutions of 0.5 mg L −1 of albendazole (corresponding to 0.055 μg g −1 in sand) (Fig. 2 ).Ov er all, our r esults indicate that albendazole interferes with pr ocesses underl ying the establishment of the symbiotic r elation-ship and impairs the capacity of plants for P uptake but has no effect on the plant host.

Albendazole affects arbuscule development and functionality
Following up on the observation that albendazole reduces the P content of plant shoots, we determined, in a second experiment, the expr ession le v els of thr ee genes known to be involved in arbuscules de v elopment, and arbuscule-mediated P and N uptake by plants .T he expr ession le v els of all thr ee marker genes wer e found significantl y r educed in the r oots of plants tr eated with a solution containing 5 mg L −1 of albendazole (corresponding to 0.754 μg g −1 measur ed concentr ation of albendazole in sand), compar ed to the control plants (Fig. 3 ).The levels of LjSbtM1 were almost 6-fold decreased, and the levels of LjPT4 and LjAMT2;2 were almost 7-fold decreased.This dramatic reduction in the transcripts of the AM marker genes denotes impair ed de v elopment and functionality of the arbuscules formed on these r oots.Tr eatment of plants with solutions of a lo w er concentration of albendazole, 0.5 mg L −1 (corresponding to 0.055 μg g −1 in sand) did not result in significant changes in the gene transcript levels, compared to the control plants (Fig. 3 ).
Ov er all, the r esults obtained fr om the molecular anal ysis confirmed our observ ations fr om the microscopic analysis; albendazole has a dir ect negativ e effect on AM symbiosis.Although the micr oscopic anal ysis did not detect impairment of the AMF root colonization when plants were treated with a solution of 5 mg L −1 of albendazole (Fig. 1 ), this was detected by the RT-q-PCR analysis and concurred with the significant reduction in P uptake by plants (Fig. 2 ) when exposed to the same concentration levels of albendazole.Based on these results we suggest that an adequate number of arbuscules are formed on the roots of the plants treated with the solution of 5 mg L −1 of albendazole, ho w e v er , these arbuscules are not well de v eloped and fully functional.Our results indicate that the presence of albendazole in the soil at concentr ation le v els that ar e expected to be found in r egularl y manur ed a gricultur al soils has toxic effects on the symbiosis of the fungus R. irregularis with the model legume L. japonicus , resulting in limitation of the fungus potential to provide the plants with essential n utrients lik e phosphorus.More in de pth anal ysis via tr anscriptomic a ppr oac hes ar e needed to establish the full extent of the response of AMF to albendazole exposure.

Albendazole dissipation
We also follo w ed the dissipation of albendazole in our system to verify the level and the duration of the exposure of the plant-AMF system to the anthelminthic compound.Albendazole sho w ed a slow dissipation in the absence of plant and AMF with more than 80% of its initially detected amount remaining in the system at the end of the study (Fig. 4 ).In contrast, in the presence of plants and AMF we noted a significant dissipation of albendazole which exceeded 50% of its initial concentration level.These results suggest that the presence of plants and AMF facilitates the r emov al of albendazole from sand.In the absence of an established biota in our sand system, beyond AMF and the roots of L. japonicus , we speculate that the gradual reduction of albendazole levels is driven either by an increasing uptake of the anthelminthic compound by plant roots or by a transformation of albendazole by AMF or by a combination of both activities .T he capacity of plants to uptake albendazole through their root system has been previously shown (Stuc hlík ov á Raisová et al. 2017 ), while the contribution of AMF in the enhancement in the degradation of organic pollutants like pesticides has been also reported (Wang et al. 2011 ).Ho w e v er, it should be stressed that the dissipation patterns of albendazole in our system are expected to differ from its environmental fate in a gricultur al soils .T he dissipation of anthelmintics, including albendazole, in soil is driven by biotic (microbial degradation) and abiotic processes (adsorption and leaching), which are less important in our system, and their r elativ e contribution to anthelminthics dissipation is affected by soil properties like pH and organic carbon content (Muta vdži ć Pa vlo vi ć et al. 2018, P orto et al. 2021, Lagos et al. 2022 ) ).

Conclusions
We provide first evidence for the toxicity of the antlhelminthic benzimidazole compound albendazole, which unlike the macrocyclic lactone ivermectin, inhibited the plant colonization capacity and function of the AMF R. irregularis with the latter constituting a more sensitive and ecotoxicologically relevant endpoint to follow.Further tests will expand our toxicity testing to the full range of anthelminthic compounds with the aim to benchmark the current assay as a tool for assessing the potential toxicity of veterinary drugs like anthelminthics to the soil microbiota.This study provides the first indication that veterinary drugs like anthelminthics may harm beneficial plant symbiotic interactions and highlight the need for consideration of k e y soil microbial groups like AMF in ecotoxicological testing of pharmaceuticals, that may r eac h the soils thr ough the use of manure and sludge as or ganic fertilizer, or thr ough irrigation of a gricultur al fields with tr eated waste water.The r esults of suc h tests ar e expected to contribute to the design of ecofriendly and safe a gricultur al pr actices in the future.

Figure 1 .
Figure 1.Arbuscular mycorrhizal fungi (AMF) root colonization levels after application of veterinary drugs.(A) Application of albendazole affects AMF root colonization.(B) Application of ivermectin does not affect AMF root colonization.Levels of AMF root colonization five weeks after the application of different concentrations of albendazole or ivermectin (or DMSO for the control treatment).Comparisons are between drug-treated and DMSO-tr eated (contr ol) plants of L. japonicus .Statistical analysis w as performed b y t-tests: * P < 0.05 (ns = not significant).No statistically significant difference was detected for invermectin treatments.(n = 5)

Figure 2 .Figure 3 .
Figure 2. Application of albendazole decreases phosphorus content in the plant shoot.Phosphorus content in the shoots of L. japonicus plants five weeks after the application of different concentrations of albenzole (or DMSO for the control treatment).Comparisons are between albendazole-treated and control plants.Statistical analysis was performed by t -tests: * * P < 0.01 (n = 4) (ns = not significant)

Figure 4 .
Figure 4. Le v els of albendazole in the plant growth substrate.Albendazole concentration was determined by HPLC analysis in sand samples from magentas boxes with or without plants and AMF.The initial concentration of albendazole in the solution was 5 mg L −1 .Statistical analysis was performed by one-way ANOVA followed by Tuk e y's post-hoc test (three technical replicates) ( P < 0,05 ).Significant differences are indicated by different letters.